Intermediate filaments (IF) are the ubiquitous constituents of the cytoskeletons of eukaryotic cells. They consist of five different types, of which the most numerous and complex are the type I and type II keratins that are widely expressed in epithelia. We are interested also in the related IF of other cell types in order to understand their roles in biology. We continued a major study in collaboration with other investigators in Switzerland, Germany and New Zealand to solve the three-dimensional structure of vimentin IF. These IF have been chosen because: (a) they are homopolymeric, and therefore likely to be somewhat simpler to solve; and (b) they have a very high sequence homology with keratin IF, and thus many of the structural principles adduced for vimentin should be applicable to keratin IF. (1) Work was completed on numerous deletion / mutation constructs which cumulatively cover the entire portion of vimentin. Chemical sequencing of these constructs have revealed interactions of the N- and C- terminal 100 amino acids with the coiled-coiled region of the molecule and revealed the role of these regions in the early (monomer to tetramer) stages of the filament assembly. The results were published. A second paper including the full filament growth from early subunits is in preparation. It may also include a refinement of the final, mature IF structure by spectroscopic (EPR) data from a collaborating laboratory (University of California at Davis) leading to completion of this study. (2) A similar project on the role of the head domain of hair keratins in the assembly of hair keratin intermediate filaments was initiated. Thirty two mutant and deletion constructs were created and the proteins have been purified. Initial experiments, with two of the constructs, revealed specific amino acid interactions between the head and rod domains. Work was suspended because of the lack of human resources. (3) Characterization of human epiplakin as a cytolinker: RNAi-mediated epiplakin depletion leads to the disruption of keratin and vimentin IF networks. Epiplakin is a member of the plakin family with multiple copies of the plakin repeat domain (PRD). We studied the subcellular distribution and interactions of human epiplakin by immunostaining, overlay assays, and RNAi knockdown. Epiplakin decorated the keratin IF network and partially that of vimentin. In the binding assays, the repeat unit (PRD plus linker) showed strong binding and preferentially associated with assembled IF over keratin monomers. Epiplakin knock-down revealed disruption of IF networks in simple epithelial but not in epidermal cells. In rescue experiments, the repeat unit was necessary to prevent the collapse of IF networks in transient knock-down; however, it could only partially restore the keratin but not the vimentin IF network in stably knocked-down HeLa cells. We infer that epiplakin is a versatile cytolinker with functions involved in maintaining the integrity of IF networks in simple epithelial cells. Furthermore, we observed an increase of epiplakin expression in keratinocytes after the calcium switch suggesting the involvement of epiplakin in the process of keratinocyte differentiation. The work was completed and submitted for publication. This project will not be further pursued in the LSB but certain ongoing lines of investigation will be continued in NIAMS by Dr L. Marekov.